Explorative study on scale cortisol accumulation in wild caught common dab (Limanda limanda)

Background Flatfish live in a diverse marine ecosystem that is changing due to natural variations and anthropogenic influences. These changes can evoke a stress response mainly resulting in production of the glucocorticoid cortisol, which mediates effects on various levels of biological organization. The finding that cortisol accumulates in fish scales, offering a retrospective view on cortisol production, provides opportunities to use this matrix for chronic stress assessment. The present study is the first to gather information on scale cortisol concentration in wild-caught common dab (Limanda limanda), based on a two-pronged approach using (1) field measurements and (2) a laboratory in vivo-study where wild-caught dab were fed by cortisol-spiked feed during 30 or 90 days to demonstrate the possible accumulation of cortisol in the scales and to evaluate its impact on fish health. Results Based on the field measurements, the average scale cortisol concentration in wild-caught fish was 0.0034 ± 0.0046 µg kg−1 scale (n = 67). This indicates that wild common dab is indeed able to incorporate cortisol in the scales. Based on the experimental data, the cortisol-fed fish showed an increased plasma cortisol concentration (80.16 ± 82.58 µg L−1) compared to the control group (4.54 ± 9.57 µg L−1) after 30 days of cortisol feeding. The increase in plasma cortisol concentration was positively correlated with an increased cortisol concentration in the scale after 30 days of cortisol-spiked feeding. This correlation was, however, no longer observed after 90 days of cortisol-spiked feeding. Interestingly, cortisol concentration of the scales on the pigmented side was significantly higher compared to the non-pigmented side. Some health parameters such as epidermal thickness, body condition and Ichthyobodo sp. parasitic infection showed a correlation with scale cortisol concentration after 30 days. Conclusions We have demonstrated that common dab is able to accumulate cortisol in its scales. This seems to occur proportionally to circulating concentrations of plasma cortisol in fish fed with cortisol supplemented feed after 30 days. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-022-03385-3.

At T30, the degree of vacuolization was negatively correlated with the PCC of the CONT fish (p = 0.0605), and a significant positive correlation was observed in the CORT group (p = 0.0092). The amount of intra-hepatocellular glycogen present in the liver, showed a significant negative correlation with PCC in the CONT group after 30 days (p = 0.0269), while SCC was not significantly correlated with the degree of vacuolization nor with the glycogen content (CONT: p = 0.2373; CORT: p = 0.4366). At T90, no significant correlations were observed, neither with the PCC nor with SCC.
In the gills, no significant correlation was observed between PCC or SCC and the total number of goblet cells neither at T30 nor at T90. No significant correlation was observed between the thickness of the epidermal tissue and the PCC, neither at T30 nor at T90 and in neither group. Thickness of the epidermal skin tissue tended to be positively associated with the SCC in CORT group at T30 (p = 0.0650), though not in the CONT fish (p = 0.8925). At T90, no significant correlations were observed.

Hematology
Detailed results of effects on hematology and the correlation with PCC and SCC are provided in Table S2.
No significant correlation was observed between PCC or SCC and the plasma osmolality, not in both groups and not at T30 nor at T90.
Hematocrit tended to be correlated with the PCC in CONT fish at T30 (p = 0.0560), but not in

Effects on organism level
Detailed results of effects on organism level and the correlation with PCC and SCC are provided in Table S3.
No correlations were observed between growth per day and PCC or SCC. No correlation was observed between PCC and the gained weight, neither at T30 nor at T90. A significant correlation was observed between the gained weight and SCC in the CORT fish at T30 (p=0.0043). The evolution of the body condition was correlated with SCC at T30 in the CORT group (p = 0.0123). No correlations were observed between feeding response and SCC or PCC.
Parasitological examination revealed that seven fish (CONT: n = 4, CORT: n = 3) had Epitheliocystis sp. in the gills. No parasites were detected on skin of the fish.
Histological examination of the gills of fish at T30 revealed the presence of Ichthyobodo sp. in seven out of nine CORT fish, distributed over the three tanks. This ectoparasitic flagellate was present in different numbers and affected gill filaments causing slight hypertrophy and 'ruffled' appearance.
Ichthyobodo sp. was not observed in gills of CORT fish at T90, neither in CONT fish at T30 and T90.
Histological examination of the internal organs at T30 and T90 revealed no remarkable or consistent pathologies in CORT or in CONT fish. The liver of two CORT fish showed signs of a pre-tumor phase as hyperplasia of hepatocytes were detected. In the kidneys, two fish (one CORT, one CONT) showed nephrocalcinosis, one CORT fish showed thyroidization of the kidney tubuli. The spleen did not show any abnormalities. Signs of myocarditis were found in one CONT fish. In the intestinal wall of nine fish (CONT (n = 4) and CORT (n = 5)) encapsulated parasites were detected.